Introduction: Kinases are molecular switches for cellular functions involved in multiple human pathogenesis, most notably cancer. There is a continuous need for soluble and active kinases for in-vitro drug discovery and structural biology purposes. Kinases remain challenging to produce using Escherichia coli, the most convenient heterologous expression system. Methods: Six strains were chosen for expression trials aiming to obtain soluble and active kinases, BL21, BL21 pLysS, Rosetta, BL21 complemented with chaperones (DnaJ/K + GroEL/ES), and ArcticExpress. Three kinases were tested for expression EGFR, MKK3 and AurKA. The target kinase was subcloned into pET15b bacterial plasmid and transformed into the tested strain. Overnight cultures were grown with appropriate selection antibiotics and subcultured next day to OD600 0.6. Expression was tested using different IPTG concentrations (1, 0.2, and 0.05 mM) at varying temperatures (37°C, 30°C, 25°C, 18°C for regular strains and 18°C, 15°C, 10°C for Arctic Express). Samples were collected after three hours for 37°C, 30°C, and 25°C and overnight for temperatures &lt; 25°C. Uninduced and induced samples were analyzed using SDS-PAGE. Optimum conditions for each strain was used to grow 1L large scale cultures from which each kinase was purified and analyzed to compare yield and activity. Results: Different strains and expression conditions were found optimal for the tested kinases.

Optimizing the expression of soluble, active human kinases using specialized E.coli strains

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.